Stabilization of neurofilament (NF) mRNAs is a critical phenomena in determining levels of NF expression, axonal size and rate of axonal conduction. By regulating NF mRNA stability, the neuron is able to establish fundamental functional parameters of its own phenotype. The stabilization of NF mRNA and increased levels of NF expression are strongly influenced by the nature of target cell innervation and, presumably, by feedback signals from the target cells to the parent neuron. The feedback signals regulating NF expression arise during a developmental timeframe in which feedback signals from the target cells are also promoting the survival and further development of the parent neuron. The latter phenomena are believed to involve growth factors produced by the target cell which interact with receptors on the parent neuron and prevent the parent neuron from undergoing apoptosis. Neurons that reach and innervate target cells acquire growth factors and survive, while neurons that fail to innervate target cells do not acquire growth factors and undergo apoptosis. Growth factors thereby enable the developing neurons to override an intrinsic program of apoptosis, as exemplified by the inability of the developing neuron to survive when separated from the target cell by nerve transection or when grown in vitro in the absence of growth factors. During the next phase of development (between 0 and 4 weeks of postnatal development), neurons lose their dependence on growth factors for survival, as exemplified by their ability to survive in vitro in the absence of growth factors as well as by their ability to survive a nerve transection (Schwartz et al., J. Neurosci. Res., 1990, 27:193-201). The weaning of neurons of their dependence on growth factors for survival reflects a change in the expression of genes regulating apoptosis, possibly due to the recruitment of a new set of genes which serve to override apoptosis in the absence of growth factors. While the identity of these anti-apoptosis genes are unknown, it is significant that they impart a vital and unique property to the neuron during the same developmental timeframe in which there is dramatic upregulation in the expression of the three NF genes (Schlaepfer, W. W. and Bruce, J., J. Neurosci. Res., 1990, 25:39-49). The dramatic increase of NF expression is due to the stabilization of NF mRNAs (Schwartz et al., J. Biol. Chem. 1992, 267:24596-24600 and Schwartz et al., Mol. Brain Res., 1994, 27:215-220) and is mediated by factors that bind to the NF mRNAs (Ca{character pullout}ete-Soler et al., J. Biol. Chem., 1998, 273:12650-12654; and Ca{character pullout}ete-Soler et al., J. Biol. Chem., 1998, 273:12655-12661). If these same factors regulate the expression of anti-apoptotic genes that maintain neuronal homeostasis in the absence of growth factors, the factors themselves would be an important vehicle by which to identify the anti-apoptotic gene products that maintain neuronal homeostasis.
The possibility that the same regulatory factors alter post-transcriptional expression of NF genes and gene products maintaining neuronal homeostasis derives from studies of motor neuron degeneration in transgenic mice bearing neurofilament transgenes. Although the pathogenesis of motor neuron degeneration due to expression of a mutant NF-L transgene (Lee et al., Neuron, 1994, 13:975-988) or overexpression of a wild-type NF-L (Xu et al., Cell, 1993, 73:23-33) or NF-H (Cote et al., Cell, 1993, 35-45) transgene in transgenic mice is presently unknown, it has been generally assumed that the neuropathic effects result from expression of NF protein by the transgene. It has been further assumed that the additional expression of NF protein by the transgene causes motor neuron degeneration by disrupting NF assembly or transport in NF-rich motor neurons (Collard et al., Nature, 1995,375:61-64; Bruijn, L. I. and Cleveland, D. W., Neuropathol. Appl. Neurobiol., 1996,22:373-387).
The interpretation that the pathogenesis of experimental motor neuron degeneration is due to alterations in protein function, however, is problematic on several grounds. While NF accumulations are prima facie evidence of disrupted NF transport, they do not indicate whether disrupted transport is a cause, rather than a result, of neuronal degeneration. Accumulation of NFs is a frequent and readily detectable pathological change that does not necessarily lead to a progressive loss of neuronal viability, even with massive accumulations of NFs in motor neurons (Eyer, J. and Peterson, A. C., Neuron, 1994, 12:389-405). Nor is there appreciable loss of neuronal viability from a marked depletion of NFs due to ablation of NF-L (Zhu et al., Exp. Neurol., 1997, 148:299-316) or medium neurofilament subunit (NF-M) (Elder et al., J. Cell Biol., 1998, 141:727-739) or a spontaneous nonsense mutation of NF-L (Yamashaki et al., Lab. Invest., 1992, 66:734-743). Finally, there is the issue of specificity, as to why a widely and abundantly expressed neuronal protein should lead to the selective degeneration of a very small subset of neurons. It is also unclear why experimental motor neuron degeneration occurs from overexpression of a mouse NF-L (Xu et al., Cell, 1993, 73:23-33) or a human heavy neurofilament subunit (NF-H; Cote et al., Cell, 1993, 73:35-46) transgene, but not from overexpression of an NF-M (Wong et al., J. Cell Biol., 1995, 130:1413-1422) or a chimeric NF-H/lacZ (Eyer, J. and Peterson, A. C., Neuron, 1994, 12:389-405) transgene.
More recently, the effects of NF expression on other models of motor neuron degeneration have been examined by cross breeding transgenic lines of mice. These studies have shown that neither the time-course nor neuropathological effects of primary sensory neuronal degeneration in (wst/wst) wasted mice or primary motor neuron degeneration in SOD-1G37R mutant mice are altered by the presence of a mutant NF-H transgene (NF-H/lacZ) causing massive maldistribution of NFs within the afflicted neurons (Eyer et al., Nature, 1998, 391:584-587). On the other hand, the additional expression from a wild-type, full-length human NF-H transgene was found to prolong the lifespan and reduce the neuropathologic effects on motor neurons of the same SOD-1G37R transgenic mice (Couillard-Despres et al., Proc. Nat'l Acad. Sci. USA, 1998, 95:9629-9630). Motor neuron degeneration by an SOD-1 transgene was also slowed by ablation of the endogenous NF-L gene, thereby markedly reducing NF expression (Williamson et al., Proc. Nat'l Acad. Sci USA, 1998, 95:9631-9636). Paradoxically, the ablation of the NF-L gene enhanced the pathological effects of the mutant SOD-1 transgene on primary sensory neurons.
The severe neuropathic effects that result from low level expression of a mutant NF-L transgene (Lee et al. Neuron, 1994, 13:975-988) contrast with the mild neuropathic effects that result from overexpression of the wild-type NF-L (Xu et al., Cell, 1993, 73:23-33) transgene, thus indicating that a mutation in the NF-L transgene markedly enhances the neuropathic effects of the transgene in transgenic mice. This mutant NF-L transgene, however, contained two separate mutations, namely, a leucine-to-proline point mutation in the rod domain of the protein and a 36 bp c-myc tag that was appended to the carboxyl terminus of the protein. The c-myc tag was added in order to mark the NF-L protein from the transgene and distinguish it from the wild-type NF-L protein encoded by the endogenous NF-L gene of the mouse. The leucine-to-proline point mutation in the rod domain was intended to create a dominant disassembling subunit that leads to the disassembly of all NFs in the cell (Gill et al. J. Cell Biol., 1990, 111:2005-2019). Although the neuropathic effects of the transgene were attributed to the point mutation in the rod domain, this interpretation was not supported by a close examination of degenerating motor neurons. Close examination showed that expression of the mutant NF-L subunit did not lead to a granular disintegration of NFs, as characteristic of the dominant disassembly phenotype (Gill et al. J. Cell Biol., 1990, 111:2005-2019), nor prevent the accumulation of assembled NFs, admixed with mutant protein, in cell bodies and dystrophic neurites of the degenerating motor neurons. The inability of the mutant protein to disrupt NFs in degenerating motor neurons indicates that the mutation does not have a dominant disassembly phenotype in vivo and that the accumulation of NFs are, most likely, the result rather than the cause of the degenerative state of motor neurons. Furthermore, the inability of the point mutation to alter assembled NFs also negates the role of this mutation in mediating neuropathic effects. Hence, there must be an alternative explanation accounting for the enhanced neuropathic effect of the mutant NF-L transgene on motor neurons of transgenic mice.
It is now believed that the enhanced neuropathic effects of the mutant NF-L transgene on motor neurons of transgenic mice (Lee et al. Neuron, 1994, 13:975-988) are not due to the point mutation in the rod domain of the protein, but rather to the second mutation created by insertion of the c-myc tag onto the carboxyl terminus of the protein. While the addition of the c-myc tag does not alter the ability of a NF protein subunit to assemble into filaments (Gill et al. J. Cell Biol., 1990, 111:2005-2019), the placement of the 36 bp c-myc tag at the junction between the coding region and 3'UTR of the NF-L cDNA generates a mutant NF-L mRNA that may have altered biological properties. The discovery that the c-myc mutation in the NF-L transcript was inadvertently inserted into the major stability determinant of the transcript supports this view (Ca{character pullout}ete-Soler et al., J. Biol. Chem., 1998, 12650-12654 and Ca{character pullout}ete-Soler et al., J. Biol. Chem., 1998, 12655-12661). Thus, the neuropathic effects are believed to be due to the c-myc mutation in the NF-L mRNA. Further, expression of the mutant NF-L mRNA, not the mutant NF-L protein, is believed to mediate the neuropathic effects of the mutant transgene in transgenic mice. This discovery has profound implications regarding the pathogenesis of motor neuron degeneration in transgenic mice and on potential treatments and cures of motor neuron diseases.
The biological effects of a c-myc mutation in the NF-L transcript and the ability of NF-L mRNA, not NF-L protein, to mediate neuropathic effects have now been determined. In these experiments, expression of the NF-L transgene with only the c-myc mutation was found to have profound disruptive effects on motor neurons of transgenic mice. Moreover, similar neuropathic changes on motor neurons were reproduced in mice bearing a transgene in which the 3'UTR and c-myc mutation of NF-L was appended to a GFP reporter protein. The latter study shows that the neuropathic effects of the mutant NF-L transgene are due to elements in the NF-L transcript, not to the expression of NF-L protein. Further, a less severe form of motor neuron degeneration was seen in mice bearing a chimeric transgene in which the NF-L 3'UTR was appended to the GFP reporter gene. These findings confirm the presence of elements in the NF-L 3'UTR with neuropathic effects on motor neurons of transgenic mice and indicate the neuropathic effects are enhanced by insertion of the c-myc mutation in the transgene. The results indicate that the milder form of motor neuron degeneration in mice bearing a wild-type NF-L transgene (Xu et al., Cell, 1993, 73:23-33) can also be attributed to neuropathic effects of an element in the NF-L 3'UTR. Finally, biochemical studies now show that similar stability determinants are present in the 3'UTR of the NF-L and NF-H transcripts, thus indicating that motor neuron degeneration from overexpression of a NF-H transgene (Cote et al., Cell, 1993, 35-45) can be attributed to the neuropathic effect of a common element in the NF-H and NF-L 3'UTRs. In summary, it has now been demonstrated that motor neuron degeneration in all three transgenic models arising from expression of different NF transgenes (Xu et al., Cell, 1993, 73:23-33; Cote et al., Cell, 1993, 35-45; Lee et al., Neuron, 1994, 13:975-988) arises from expression of NF transcripts. Further, the neuropathic effects of NF transcripts are due to cis-acting elements that regulate the stabilities of the transcripts and bind common trans-activating factors.
The identification of common cis-acting elements in the 3'UTR of NF mRNAs that lead to motor neuron degeneration in transgenic mice provides important evidence relating to pathways and components thereof which mediate the neuropathic effects. A prime candidate component is the ribonucleoprotein (RNP) complex in brain extracts which binds to the cis-acting element in the 3'UTR of NF-L and NF-H mRNAs that stabilize the transcripts and confer neuropathic effects in transgenic mice. The RNP complexes contain a novel 43 kDa neurofilament mRNA binding protein, referred to as NFRBP-1, that also binds directly to the cis-acting element in the 3'UTR of NF-L and NF-H mRNAs. Insertion of the c-myc mutation into the stability determinant of the NF-L transcript alters the binding of RNP complexes and NFRBP-1 to the stability determinant and disrupts the ability of the stability determinant to regulate the stability of the transcript. Thus, the binding of NFRBP-1 to the NF transcript is closely associated with the stabilization of the transcript and with the enhanced neuropathic effects on motor neurons of transgenic mice that result from presence of the c-myc mutation in the NF-L transgene. The identification of NFRBP-1 and its interactions with other components of neuronal cells and tissue are believed to represent a key link in the pathways leading to motor neuron degeneration in transgenic mice and to motor neuron disease.